DNA damage accumulates during life and is thought to contribute to the aging and genomic instability. Therefore, defining those proteins and pathways that maintain genomic instability may be critical in preventing aging and age-related degeneration. This project aims to understand what roles the five human RecQ helicases play in DNA repair and genomic stability. We and other groups find that the human RecQ helicases participate in DNA repair, and more specifically in certain subpathways of DNA repair. We find that the RECQL4 protein participates in DNA repair of double and single strand breaks and that cells from individuals with deficient RECQL4 are defective in DNA repair. Using real time imaging, we have shown that RECQL4 is recruited to sites of double strand breaks and that its retention kinetics are different than WRN or BLM. Additionally, we mapped the domain of RECQL4 necessary for DNA damage recruitment. Biochemically, we are characterizing the RECQL4 protein and, while its helicase function is in many ways similar to WRN and BLM helicases, there are also significant differences. We have shown that RECQL4 has a very limited substrate range and that its helicase activity can be seen on short fork DNA substrates but not on long DNA substrates. Mutations in RECQL4 are associated with three diseases Rothmund Thomson, RAPADILINO and Baller-Gerold syndrome. Using site-directed mutagenesis and recombinant proteins, we are characterizing the biochemical properties of RECQL4 patient-associated mutations. Consistently we are finding that patient mutations associated with RAPADILINO syndrome show loss of helicase but not strand exchange activity. Whereas mutations associated with RTS render the protein unstable. We are also exploring potential protein interactions and protein complexes that RECQL4 participates in. Previously we showed that RECQL4 could modulate core BER proteins like APE1, pol B; and FEN1. More recently our work has focused on defining the role of RECQL4 after DSB induction and during telomere maintenance. We find that RECQL4 loss increases telomeric sister chromatid exchanges. Furthermore, we discovered that RECQL4 specifically interacts with telomeric proteins. Thus, we are continuing to define and explore the catalytic properties of RECQL4, potential protein:protein interaction partners especially following DSBs and post-translational modifications of RECQL4. We recently reported that RECQL4 localizes to mitochondria where it may play a role in mtDNA maintenance. Future work will explore in what capacity RECQL4 functions in mtDNA replication and/or transcription. RECQL5 is another member of the RecQ family for which little information is available. We have previously reported that WRN, BLM and RECQL4 can stimulate core base excision repair proteins. Thus we evaluated whether RECQL5 could also stimulate and interact with BER proteins. Indeed, we found that RECQL5 could interact with and stimulate FEN1 incision reactions. We have now expanded the analysis of RECQL5 in BER by performing a microarray analysis and find numerous DNA repair related genes differentially regulated upon RECQL5 loss. Therefore the RecQ helicases not only regulate the activities of BER proteins but they also have the capacity to modify BER gene expression patterns. In addition to RECQL5s role in BER, we are evaluating its role in DSB repair. Like the other RecQ helicases, we find that RECQL5 is recruited to DSBs but its retention kinetics are different than the other RecQ helicases. Additionally, we mapped the domain of RECQL5 that is necessary for DSB recruitment. Thus, in the DSB damage response, it appears that the various RecQ helicases have both complementary and independent roles. Additionally, we have recently shown that RECQL5 physically and functionally interacts with Topoisomerase II alpha. This interaction stimulates the decatenation activity of Topoisomerase II. Consistent with these observations, metaphase spreads generated from RECQL5-depleted cells exhibited undercondensed and entangled chromosomes. Furthermore, RECQL5-depleted cells have an activated a G2/M checkpoint and undergo apoptosis. These phenotypes are similar to those observed when Topoisomerase II catalytic activity is inhibited. These results reveal an important role for RECQL5 in the maintenance of genomic stability. &#8195;